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Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.
MedLine Citation:
PMID:  23280612     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization - mass spectrometric analysis of the affinity - elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley & Sons, Ltd.
Authors:
Gabriela Paraschiv; Cécile Vincke; Paulina Czaplewska; Marilena Manea; Serge Muyldermans; Michael Przybylski
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of molecular recognition : JMR     Volume:  26     ISSN:  1099-1352     ISO Abbreviation:  J. Mol. Recognit.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-02     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9004580     Medline TA:  J Mol Recognit     Country:  England    
Other Details:
Languages:  eng     Pagination:  1-9     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 John Wiley & Sons, Ltd.
Affiliation:
Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, 78457, Konstanz, Germany.
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